Proc Natl Acad Sci U S A 94(17): 8948-8953

Stimulation of neurite outgrowth using an electrically conducting polymer

^{Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; and}^{Department of Surgery, Children’s Hospital, Harvard University, Boston, MA 02115}

^{Present address: Department of Chemical Engineering, University of Texas, Austin, TX 78712.}

^{C.E.S. and V.R.S. contributed equally to this work.}

^{To whom reprint requests should be addressed. e-mail:}ude.tim@regnalr.

Contributed by Robert Langer

Accepted 1997 Jun 13.

Abstract

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (n = 5643) compared with 9.5 μm (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).

Keywords: conductive polymer, oxidized polypyrrole, nerve regeneration, PC-12 cells, electrical stimulation, neuronal stimulation

Abstract

The current clinical approach to repair a peripheral nerve over a gap involves the utilization of autologous nerve grafts (1). However, this procedure has several disadvantages, including loss of function at the donor nerve graft site and mismatch of damaged nerve and graft dimensions. As an alternative to nerve autografts, natural and synthetic tubular guidance channels have been the subject of intensive research (2). Guidance channels help direct axons sprouting off the regenerating nerve end, provide a conduit for diffusion of neurotrophic and neurotropic factors secreted by the damaged nerve stump, and minimize infiltrating fibrous tissue. Materials that have been investigated for nerve repair range from autologous veins (3) and muscles (4), to prosthetic tubes derived from collagen, laminin, fibronectin (5–7), silicone (8), and various other biodegradable and nonbiodegradable polymers (2). However, the engineering of an ideal nerve guidance channel that provides an attractive clinical alternative to nerve autografts remains a challenge.

Past work has demonstrated that electrical charges play an important role in stimulating either the proliferation or differentiation of various cell types. For example, it has been shown that neurite outgrowth is enhanced on electrets such as poled polyvinylidene fluoride (9, 10) and poled polytetrafluoroethylene (11). Electrets are broadly defined as materials possessing quasi-permanent surface charge because of trapped monopolar charge carriers. In piezoelectric materials such as poled polyvinylidene fluoride, transient surface charges are generated as a result of minute mechanical deformations of the material, whereas poled polytetrafluoroethylene displays a static surface charge. Enhanced neurite outgrowth has been attributed to the presence of these surface charges. Furthermore, extensive research both in vitro and in vivo has shown that electromagnetic fields play an important role in neurite extension and regeneration of transected nerve ends (12–14).

It occurred to us that a polymer scaffold or guidance channel derived from an electrically conducting polymer could prove potentially useful in not only providing neuronal guidance but in localizing electromagnetic stimulation as well. The electrically conducting polymer, oxidized polypyrrole (PP), was chosen for this study because of its inherent electrical conductive properties, ease of preparation, flexibility of altering surface characteristics, and its in vitro compatibility with mammalian cells (15, 16). In its oxidized form, polypyrrole is a polycation with delocalized positive charges along its highly conjugated backbone and is an electronic conductor (17). Charge neutrality is achieved by the incorporation of negatively charged ions termed “dopants.” PP also exhibits reversible electrochemistry allowing for the control of surface-charge density by varying the oxidation state of the polymer. For example, the application of a reduction potential to PP converts it from a conductive form to its neutral (insulative) form with the release of dopant anions or the incorporation of cations to maintain charge neutrality. Potential biomedical applications have been developed based on the electrochemical properties of PP. These include the use of PP as a matrix for controlled delivery of dopamine (18) and as a biosensor for detection of glucose (19) or proteins (20).

Surface characteristics such as charge density and wettability play a key role in protein adsorption and cell–substrate interactions. Recently, it has been shown that both cell–surface interactions and cellular functions (e.g., DNA synthesis) on PP thin films can be controlled by either changing the oxidation state of the polymer (15) or by changing the wettability (hydrophobicity) of the polymer film using appropriate dopants (16). Surface characteristics are critical because the interaction of endogenous proteins with a biomaterial at the implantation site will have a significant impact on the adhesion, differentiation, and proliferation of surrounding cells. Flexibility at the level of controlling protein adsorption provides a means to engineer materials that yield predictable and desirable cell–surface interactions. Therefore, PP is an interesting candidate for tissue engineering applications, by virtue of its inherent electrical conductivity, the ease with which one can control crucial surface properties such as wettability and charge density, and its compatibility with mammalian cells.

In this study, the utility of the electrically conductive polymer, PP, as a substrate to enhance nerve cell differentiation in culture was evaluated with the hope of ultimately using electrically conducting polymers to stimulate in vivo nerve regeneration. We show that PP is a suitable material for in vitro nerve cell culture and that application of an electric stimulus through PP enhances neurite outgrowth. We also show that PP does not elicit an adverse tissue response when implanted in rats.

Acknowledgments

We thank N. Chen, I. George, R. Bellamkonda, P. Basser, D. Odde, T.-H. Kim, R. Solomon, I. Joris, M. Frangelo, and S. Charnick for their help. This work was supported by a National Science Foundation Grant (BES-9525913) to R.L. and a National Institutes of Health postdoctoral fellowship to C.E.S.

Acknowledgments

ABBREVIATIONS

PP	polypyrrole
PLA	poly(l-lactic acid)
PLGA	poly(lactic acid-co-glycolic acid)
TCPS	tissue culture polystyrene
ITO	indium tin oxide
NGF	nerve growth factor

ABBREVIATIONS

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